Gliding motility assay using 5528mM D2O in solution. Replicate 2/3. ROI 14/15.

• This is a gliding motility assay using kinesin-1 and rhodamine labeled microtubules.• The buffer used was PEM (also known as BRB80) with the inclusion of 5528mM D2O.• Images were taken with an Andor Luca EMCCD camera on an Olympus IX-71 microscope using a 60x 1.42NA PlanApo objective.• This fileset is replicate 2/3 and ROI 14/15.• Images were taken to investigate speed variations of microtubules due to the inclusion of D2O in the motility assay.• The temperature of the slide was held constant at 32°C by an objective heater. Early ROIs, i.e. 1-5, have slower giding speeds due to the increasing temperature of the slide which, was prepared at room temperature before observation. After ROI 5 the gliding speed stabilizes.• Pixels lengths were measured to be 166nm/pixel using a stage micrometer.• After the files have downloaded, a simple command line entry will convert the images to a movie. Mencoder must be installed. ls > list.txt | mencoder mf://@list.txt -mf w=496:h=658:fps=30:type=png -ovc lavc -lavcopts vcodec=mpeg4:mbd=2:trell -o output.avi• Upload of these files were made possible through the figshare API.

• 本实验为基于驱动蛋白-1（kinesin-1）与罗丹明标记微管的滑行运动测定实验。
• 实验所用缓冲液为PEM（又称BRB80），并添加了5528mM重水（D₂O）。
• 图像采集采用搭载60×、1.42数值孔径（NA）PlanApo物镜的奥林巴斯（Olympus）IX-71显微镜，搭配Andor Luca EMCCD相机完成。
• 本次文件集对应第2/3组重复实验与第14/15号感兴趣区域（Region of Interest, ROI）。
• 本次图像采集旨在探究滑行运动测定体系中，因添加重水所引发的微管滑行速度变化。
• 通过物镜加热器将载玻片温度恒定维持在32℃。早期采集的感兴趣区域（即1-5号）因载玻片于观测前在室温下制备，其温度随时间逐渐升高，导致微管滑行速度偏慢；第5号感兴趣区域之后，滑行速度趋于稳定。
• 使用载物台测微尺（stage micrometer）标定得到像素长度为166nm/像素。
• 文件下载完成后，可通过简单的命令行指令将图像批量转换为视频。需预先安装Mencoder工具，具体执行指令为：ls > list.txt | mencoder mf://@list.txt -mf w=496:h=658:fps=30:type=png -ovc lavc -lavcopts vcodec=mpeg4:mbd=2:trell -o output.avi
• 本次文件上传通过figshare应用程序编程接口（Application Programming Interface, API）完成。